This invention is directed at antifungal synergistic combinations of fungal cell wall degrading enzyme and fungal cell membrane affecting fungicide and use thereof for topical or internal application in agriculture or medicine to inhibit germination or growth of fungi.
The primary methods of controlling disease-causing fungi on crop plants and on animals, including humans, comprise treatment with synthetic chemical pesticides. However, the exposure of man and habitats to increasing amounts of pesticides has come under criticism, resulting in a search for environmentally safer methods including the use of synergistic combinations of fungicides to reduce the amounts of application.
Poulose, A. J., in Koeller, W., ed., Target Sites of Fungicide Action, CRC Press, Boca Raton, Fla., 1992, at pages 313-314 reviews the disclosures of a number of authors directed to synergistic interaction of different lytic enzymes produced by a variety of microorganisms with a small number of, antifungal compounds including amphotericin B, benomyl, polyoxin B, kitazin P and nikkomycin.
It is an object of this invention to expand the range of synergistic combinations of fungicide/enzyme.
In one embodiment, the invention is directed to a system for inhibiting the germination or growth of a fungus, said system comprising (a) fungal cell wall degrading chitinolytic or glucanolytic enzyme; (b) antifungal fungal cell membrane affecting compound which-is not expressed by the same organism as the fungal cell wall degrading enzyme in nature, in an amount to provide a concentration where it provides about 4 to 95% inhibition of spore germination when used without (a); the weight ratio of (a) to (b) being 0.005:1 to 500,000:1.
Preferably the fungal cell wall degrading, chitinolytic or glucanolytic enzyme is present in an amount to provide a concentration where said enzyme provides 2 to 50% inhibition of spore germination when it is used without antifungal fungal cell membrane affecting compound and the antifungal fungal cell membrane affecting compound is present in an amount where it provides 10 to 70% inhibition of spore germination when it is used without fungal cell wall degrading chitinolytic or glucanolytic enzyme and the total of the percentage inhibitions provided by the fungal cell wall chitinolytic or glucanolytic enzyme and the antifungal fungal cell membrane affecting compound when each is used without the other is less than 100%.
Very preferably, the fungal cell wall degrading chitinolytic or glucanolytic. enzyme is present in an amount to provide a concentration where said enzyme provides 5 to 20% inhibition of spore germination when it is used without antifungal fungal cell membrane affecting compound and the antifungal fungal cell membrane affecting compound is present in an amount to provide a concentration where said compound provides 15 to 60% inhibition of spore germination when it is used without fungal cell wall degrading enzyme.
The term xe2x80x9csystemxe2x80x9d is used because the fungal wall degrading chitinolytic or glucanolytic enzyme and antifungal fungal cell membrane affecting compound can be applied as part of the same composition or can be applied concurrently as separate compositions or can be applied separately at different times. Preferably, the two kinds of antifungal components of the system are applied in the same composition or concurrently as separate compositions or the antifungal fungal cell membrane affecting compound is applied up to 8 hours after the cell wall degrading chitinolytic or glucanolytic enzyme.
The term xe2x80x9cinhibitxe2x80x9d is used herein to mean reduce the growth and/or development of fungi compared to where inhibiting agent is not present.
The term xe2x80x9cfungal cell wall degrading chitinolytic or glucanolytic enzymexe2x80x9d is used herein to mean chitinolytic or glucanolytic enzyme that effects lysis of fungal cell walls.
The term xe2x80x9cantifungal fungal cell membrane affecting compoundxe2x80x9d is used herein to mean sterol synthesis inhibiting fungicide, antifungal peptide antibiotic, zeamatin and proteins that are serologically related to zeamatin and antifungal lipid lytic enzymes. The term xe2x80x9cfungal cell membranexe2x80x9d means plasmalemma and membranes surrounding secretory vesicles, vacuoles, mitochondria, endoplasmic reticulum, and nuclei.
The limitation xe2x80x9cwhich is not expressed by the same organism as the fungal cell wall degrading enzyme in naturexe2x80x9d is to exclude combinations which occur in nature.
The concentration where fungal cell wall degrading enzyme individually provides a specified percentage fungal inhibition or where antifungal fungal cell membrane affecting compound individually provides a specific percentage fungal inhibition can be determined as follows: Assays are performed under sterile conditions. Equal volumes of spore suspensions, 3xc3x97 potato dextrose broth, and the test solution or suspension in 5 mM Tris-HCl (pH 6.0) or 5 mM potassium phosphate buffer (pH 6.7) are mixed. The control is the same as the test solution except for the control the antifungal agent is omitted. The assay mixtures (total volume 45 or 30 xcexcl) are incubated on flat-bottomed ELISA plates, each containing 96 wells, with 2,000 to 3,000 spores per well, at 25xc2x0 C. After 22 to 30 hours, the plates are placed under an inverted microscope. The percentage of conidia germinating is determined as the percentage of the first 100 spores randomly found in a well. In addition, the lengths of 20 germ tubes are measured and averaged. All experiments are performed twice, with three replicates for each treatment. The inhibition values obtained in the two experiments are combined and averaged, and standard deviations are calculated from the 6 data points. The values obtained for the control are taken as 0% inhibition and all other values are divided by the values obtained for the control and multiplied by 100% to obtain percent inhibition. Determination of concentration corresponding to a particular percent inhibition is carried out by subjecting dose-response curves to regression analysis by using a binomial regression of the third order, with R2 ranging between 0.95 and 0.99.
The system of the invention can optionally contain as additional components, for example, antifungal polyene macrolide antibiotic, antifungal epithiodiketopiperazine antibiotic, fungal cell wall biosynthesis inhibitor (e.g., chitin synthetase inhibitor or xcex2-1,3-glucan synthetase inhibitor) and/or detergent, in an inhibition improving amount. The term xe2x80x9cinhibition improving amountxe2x80x9d is used to mean an amount causing a greater % fungal inhibition than if the additional component(s) is/are omitted.
In another embodiment, the invention is directed to a method of inhibiting the germination or growth of a fungus and comprises contacting such fungus or a locus to be protected from such fungus with an antifungal effective amount of combination of fungal cell wall degrading chitinolytic or glucanolytic enzyme in a concentration where said enzyme individually provides 2 to 50% inhibition of spore germination and antifungal fungal cell membrane affecting compound which is not chitinolytic or glucanolytic enzyme and which is not expressed by the same organism as the fungal cell wall degrading enzyme in nature in a concentration where said compound individually provides about 4 to 95%, for example, 10 to 70%, inhibition of spore germination, the total of the percentage inhibitions individually provided by the fungal cell wall degrading chitinolytic or glucanolytic enzyme and the antifungal fungal cell membrane affecting compound being less than 100%.
The term xe2x80x9clocus to be protected from such fungusxe2x80x9d includes seeds, roots, stems, leaves, flowers and fruits to be protected and to the soil surrounding seeds and roots to be protected, as well as animal or. human tissues or organs to be protected.
In another embodiment, the invention is directed to a method of protecting from a fungus, a plant which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme at a level of about 0.05 to 5% of total cellular protein, said method comprising contacting said plant with an antifungal effective amount of an antifungal fungal cell membrane affecting compound at a concentration where it individually provides about 4 to 95%, for example, 10 to 70% inhibition of spore germination.
In still another embodiment, the invention is directed to a transgenic plant protected against pathogenic fungi which is a plant susceptible to fungal attack which has been transformed to contain gene which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme at a level of about 0.05 to 5% of total cellular protein and also which has been transformed to contain gene which expresses protein antifungal cell membrane affecting compound or which has been infected with transgenic endomorphic microorganism producing said protein antifungal fungal cell membrane affecting compound typically in the xylem, in an amount to provide a concentration of said compound where it individually provides about 4 to 95% inhibition of spore germination.
The term xe2x80x9cantifungal effective amountxe2x80x9d is used herein to mean an amount effective to inhibit the germination or growth of a fungus.